Effects of calpain inhibition on IkBα degradation following PBMC activation: identification of calpain cleavage sites

Abstract

We previously demonstrated calpain‐mediated activation of PBMCs. In order to examine whether calpain plays a role in the degradation of IkBα and NFkβ translocation in the mechanism of T cell activation, human PBMCs were activated using anti‐CD3/CD28 (HIT3A/CD28.2). Degradation of IkBα was monitored by Western blot, degradation products were sequenced, and calpain‐cleaved sites were determined. The activated cells degraded IkBα, the inhibitor of NFkβ, relative to non‐activated cells. IkBα degradation was progressive and inhibited by calpain inhibitor calpeptin in a dose and time dependent manner. Further, calpain inhibition increased IkBα levels above those observed in non‐activated negative controls. Recombinant IkBα was incubated with purified porcine mcalpain to initiate the degradation of IkBα. The degradation products were separated via SDS–PAGE and visualized using Coomassie Stain. The stained degradation products were isolated and subjected to sequencing. Calpain cleaved the recombinant IkBα most effectively when incubated with IkBα at a ratio of 1 : 1 for 20 min in the presence of 0.1% Triton‐X100. After incubation, several degradation products were detected, including a peptide corresponding to a cleavage of IkBα between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire Signal Response Domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. These results show that calpain plays an important role in IkBα degradation, a crucial event in T cell activation and is involved in the normal turnover of IkBα.Acknowledgements: Supported by grants from NIH‐NINDS.